Ammonia is the principal nitrogenous waste product of teleosts and many invertebrates in both freshwater and seawater. The ammonia results from the deamination or transamination of proteins the organism receives via its diet. However, high ammonia concentrations can be toxic to many of these same aquatic organisms. In natural systems, such as lakes, rivers and oceans, the concentration of ammonia rarely reaches deleterious levels because the density of fish (and other organisms) per mass of water is low.
However, in man-made aquatic systems such as aquaculture rearing pens, tanks, raceways and ponds plus aquaria, both public and private, ammonia can reach toxic concentrations, sometimes very quickly. One reason for this is that in the above-named systems the fish density can be very large in relation to the small amount of water. Another reason is that in many of these systems the water is not continually changed; rather it recirculates through the system with only periodic partial water changes.
Therefore, most aquaculture systems and aquaria use filtration, in one form or another, to maintain a degree of water quality that is suitable for the maintenance and growth of aquatic organisms. A major component of any such filtration unit is the biological filter. The biological filter gets its name from the fact that it acts as a substrate or site for the growth of bacteria which have the capability to convert, by way of oxidation, ammonia to another compound—nitrite. High concentrations of nitrite can also be toxic but there are other species of bacteria which grow on the biological filter and oxidize the nitrite to nitrate, such as those described in U.S. Pat. Nos. 6,268,154, 6,265,206 and 6,207,440, each of which is incorporated by reference herein in its entirety as if fully set forth. Nitrate is considered non-toxic to aquatic organisms except in extreme cases of very high concentrations.
There are other situations or applications which use biological filters. These include sewage treatment facilities, wastewater treatment facilities and drinking water filtration plants. While each will have its own particular reason for using a biological filter, the goal is the same: the conversion of toxic inorganic nitrogen compounds to less harmful inorganic nitrogen substances. Biological filtration is necessary for many facilities to meet the National Recommended Water Quality Criteria as set by the Environmental Protection Agency (EPA) of the United States of America.
The oxidation of ammonia to nitrite is a process mediated by ammonia-oxidizing bacteria (AOB). Specifically, it is a two step oxidation process involving the conversion of ammonia to nitrite according to the following equations:NH3+O2+H2O+2e−->NH2OH+H2O  (1)NH2OH+H2O ->NO2−+5H++4e−  (2)
The oxidation of nitrite to nitrate is also a bacterially-mediated process. Specifically, it is a one step oxidation process involving the conversion of nitrite to nitrate according to the following equation:NO2−+H2O ->NO3−+2H++4e−  (1)
The most commonly studied nitrite oxidizing bacteria (NOB) is Nitrobacter winogradskyi. It was originally isolated from soils and is purported to be the active NOB in aquaculture facilities (Wheaton, F. W. 1977. Aquacultural Engineering. John Wiley & Sons, Inc. New York.), in wastewater treatment facilities (Painter, H. A. 1986. Nitrification in the treatment of sewage and waste-waters. In Nitrification J. I. Prosser ed. IRL Press. Oxford.) and in aquaria (Spotte, S. 1979. Seawater Aquariums—The Captive Environment. Wiley-Interscience. New York). These references, and all other references cited herein are hereby incorporated by reference in their entirety as if fully set forth.
However, recent research conducted with modern molecular methods which use the uniqueness of the DNA sequence of an organism (or group of organisms) has shown that N. winogradskyi and its close relatives were below detection limits in freshwater aquaria environments (Hovanec, T. A. and E. F. DeLong. 1996. Comparative analysis of nitrifying bacteria associated with freshwater and marine aquaria. Appl. Environ. Microbiol. 62:2888-2896.). Furthermore, research has shown that bacteria from the phylum Nitrospira are responsible for the oxidation of nitrite to nitrate in aquaria (Hovanec, T. A., L. T. Taylor, A. Blakis and E. F. DeLong. 1998. Nitrospira-like bacteria associated with nitrite oxidation in freshwater aquaria. Appl. Environ. Microbiol. 64:258-264.) and in wastewater treatment facilities (Burrell, P. C., J. Keller and L. L. Blackall. 1998. Microbiology of a nitrite-oxidizing bioreactor. Appl. Environ. Microbiol. 64:1878-1883.). However, the Nitrospira isolate determined to be responsible for nitrite oxidation in freshwater aquaria was not found in marine aquaria (Hovanec et. al. 1998).
Nitrospira marina was first discovered by Watson in 1986 (Watson, S. W., E. Bock, F. W. Valois, J. B. Waterbury, and U. Schlosser; 1986. Nitrospira marina gen. nov., sp. nov.: A chemolithotrophic nitrite oxidizing bacterium. Archives Microbiology, 144:1-7). However, it was not considered an important or dominant nitrite-oxidizing organism in natural (soils, marine or freshwaters nor reservoirs) or artificial environments (wastewater treatment facilities) (Abeliovich, A. 2003. The Nitrite Oxidizing Bacteria. In M. Dworkin et al. Eds. The Prokaryotes: An Evolving Electronic Resource for the Microbiological Community, third edition, release 3.13, March 2003. Springer-Verlag, New York). A second species of Nitrospira (Nitrospira moscoviensis) was isolated from a partially corroded iron pipe in a heating system of a building located in Moscow, Russia. This bacterium grew optimally at 39° C. in a non-marine medium (Abeliovich, A. 2003). It has also been reported that the microbial consortium of a marine moving bed reactor (MBB) included both AOB (Nitrosomonas cryotolerans) and NOB (Nitrospira marina), along with a number of heterotrophic bacteria. (Y. Tal, J. E. M. Watts, S. Schreier, K. R. Sowers and H. J. Schreier, 2003. Characterization of the microbial community and nitrogen transformation process associated with moving bed bioreactors in a closed recirculated mariculture system. Aquaculture 215 (2003) 187-202.)
An environmental factor of particular import with aquaria environments and wastewater treatment is salinity, and, more specifically, the numerous physicochemical differences between freshwater and saltwater environments. The distinction among various NOB in their ability to tolerate such dramatic changes in local environment is critical in the design of these systems and implementation of NOB therein. As such, a demonstrated tolerance by a particular NOB to a saltwater environment may render that NOB suitable for use in particular aquaria and wastewater environments. Moreover, an ability to withstand the change between a freshwater and saltwater environment may have even broader implications, such as suitability of a particular NOB for use in a range of environments, both freshwater and saltwater.
Furthermore, the storage and transport of NOB is often limited to liquid and similar, potentially inconvenient media, owing, at least in part, to the inability of various strains of NOB to withstand a freeze-drying process. Freeze-drying allows one to formulate a volume of NOB into a solid, freeze-dried powder or similar composition that may be tolerant of greater fluctuations in, e.g., temperature, and may be correspondingly more practical for purposes of shipping and handling in a commercialized product and for maintaining an extended shelf-life.
Thus, there exists a need in the art for the identification of NOB which are capable of tolerating a saltwater environment and/or both saltwater and freshwater environments. There is also a need in the art for NOB that remain viable after being subjected to a freeze-drying process.